The objective of this study is to elucidate the role of the hereditary hemochromatosis gene (HFE) - Transferrin receptor (TFR1) interaction in vivo. HFE is a major histocompatibility complex (MHC) class l-like gene that forms a protein-protein complex with TFR soon after synthesis and is transported to the cell membrane. This interaction causes a conformational change in TFR1, reducing the affinity of the receptor for holo-transferrin (Fe-TF). Mutations in HFE disrupt its conformation and interaction with p2-microglobulin, and cause a decrease in cell surface expression of the HFE protein. Previous studies have investigated the HFE/TFR interaction interface by co-crystallization of HFE and TFR1, by site-directed mutagenesis and by in vitro binding studies. We chose to introduce two mutations into the murine Tfr1, L622A and R654A, to study the role of the Hfe/Tfr1 interaction in vivo. The L622A mutation disrupts a leucine residue that is essential for interaction between Hfe and Tfr1, but is not crucial for the Tfr1/Tf interaction. Conversely, the R654A substitution greatly decreases the affinity of Tfr1 for Tf but has no impact on the Hfe/Tfr1 interaction. Several parameters will be analyzed to define the phenotype of the genetically engineered mice. Liver iron stores will be used as an index of intestinal iron absorption, and serum transferrin saturation and spleen iron stores will be used to assess macrophage iron recycling and stores. The resulting data will give new insight into the function of the Hfe/TfM complex and aid in distinguishing among competing models for how Hfe regulates iron homeostasis. The specific aims of this proposal are: 1) to generate mouse models where Tfr1 is unable to interact with Tf, but its ability to bind to Hfe is unaffected, 2) to generate a mouse model where Tfr1 is able to interact with Tf, but its ability to bind to Hfe has been abrogated, and 3) to generate transgenic mouse models to examine putative signaling portions of the Hfe molecule. Knowledge gained through this study will add to our understanding of human iron disorders including hereditary hemochromatosis, iron-limited anemia, and anemia caused by chronic disease. It may inform new therapeutic strategies. The KO1 award will allow the candidate to undertake an extensive mentorship with Dr. Nancy Andrews. During this period he will have the opportunity to increase the technical skills and the knowledge base necessary for the successful generation of both transgenic and gene-targeted mice. The candidate will also learn to effectively employ this information and skill set to test complex hypotheses in iron metabolism and homeostasis. He will also have ample opportunities to interact and collaborate with leading investigators in the field through attendance at meetings and the writing of articles for publication. Most importantly, this award will assist in the transition of the candidate to become a fully independent investigator in the field of iron metabolism.